Visit www.lifetechnologies.com/transfection or contact Technical Support for specialized transfection protocols (including cell-type specific advice on use of PLUS™ Reagent and antibiotics, and a protocol for vector-based RNAi).

We recommend Opti-MEM® I Reduced Serum Medium (Cat. no. 31985-062) to dilute the DNA and Lipofectamine® LTX Reagent before complexing.

Maintain the same seeding conditions between experiments. Transfection can be performed both in the presence or absence of serum. Test serum-free media for compatibility with Lipofectamine® LTX Reagent.

Optimizing Transfections

To obtain the highest transfection performance, perform optimization by using a range of DNA and Lipofectamine® LTX amounts with and without the PLUS™ Reagent as described below for a 24-well format. For other formats, adjust the amounts according to Scaling Up or Down Transfections (below). Consider varying cell density for further optimization. Note: Visit www.lifetechnologies.com/transfection for cell-specific transfection protocols.

Standard Protocol

Use these guidelines to transfect DNA into mammalian cells in a 24-well format after optimizing the reactions as described above. If using the PLUS™ Reagent, start with a 1:1 ratio of DNA (μg) to PLUS™ Reagent (μl). All amounts are given on a per well basis. Note: Read Important Guidelines for Transfection (above) before using antibiotics.

Adherent cells: One day before transfection, plate cells in 500 μl of growth medium so that the cells will be 50–80% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes, plate 100,000–250,000 cells in 500 μl of growth medium.

High-Throughput Protocol

Use these guidelines for high-throughput transfections, or if dispensing small amounts of reagents. Pre-dilute th reagents first, and then add this largerAvolume to the diluted DNA. Discard unused diluted reagents, as diluted lipid loses activity after 5 minutes. All amounts are given on a per well basis for the 96-well format; for other formats, refer to Scaling Up or Down Transfections (below). For best results, optimize transfections as described in Optimizing Transfections

Note: Review Important Guidelines for Transfection (above) before using antibiotics.

Adherent cells: One day before transfection, plate cells in 100 μl of growth medium so that the cells will be 50–80% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes, plate 20,000–50,000 cells in 100 μl of growth medium without antibiotics

Mix Lipofectamine® LTX gently. Prepare a “Master Mix” by making a 1:10 dilution of Lipofectamine® LTX in Opti-MEM® I Reduced Serum Medium. Aliquot the appropriate amount of the “Master Mix” (equivalent to the optimized amount of Lipofectamine® LTX), and bring up the final volume to 10 μl per well with Opti-MEM® I Reduced Serum Medium. Mix gently.

Add the diluted Lipofectamine® LTX to the diluted DNA. Mix gently. Proceed to the next step within 5 minutes

Generating Stable Cell Lines

Scaling Up or Down Transfections

To transfect cells in different tissue culture formats, vary the amounts of DNA, Lipofectamine® LTX, PLUS™ Reagent cells, and medium used in proportion to the relative surface area, as shown in the table below and given on a per well basis. Determine the optimal amount required for Lipofectamine® LTX, plasmid DNA, and PLUS™ Reagent (if used) as described in Optimizing Transfections for 24-well format.

Culture vessel

Surface area per well 1

Volume plating medium

Volume dilution medium 2

Relative Surface Area (compared to 24-well)

96-well

0.3 cm2

100 μl

20 μl

0.2X

48-well

1.0 cm2

200 μl

40 μl

0.4X

24-well

2 cm2

500 μl

100 μl

1X

12-well

4 cm2

1 ml

200 μl

2X

6-well

10 cm2

2 ml

500 μl

5X

1 Surface areas may vary depending on the manufacturer.

2 Lipofectamine® LTX and DNA are diluted separately in the High Throughput Protocol, each into one half of the total volume of dilution medium.

Reverse Transfection

You may perform rapid 96-well plate transfections by plating cells directly into the transfection mix. Prepare complexes in the plate and directly add cells at twice the cell density as in the standard protocol in a 100 μl volume. Cells will adhere as usual in the presence of complexes. Optimize lipid doses, as in most cases more lipid is required for optimal transfection using this method.

Purchaser Notification

This product is covered by Limited Use Label License No.5: Invitrogen Technology (see the Invitrogen catalog or our website, www.lifetechnologies.com). By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses.